Methods and Compositions for Treating Conditions of the Eye

ABSTRACT

Provided are methods and compositions for the photodynamic therapy (PDT) of ocular conditions characterized by the presence of unwanted choroidal neovasculature, for example, neovascular age-related macular degeneration. The selectivity and sensitivity of the PDT method can be enhanced by combining the PDT with an anti-angiogenesis factor, for example, angiostatin or endostatin, or with an apoptosis-modulating factor. Furthermore, the selectivity and sensitivity of the PDT may be further enhanced by coupling a targeting moiety to the photosensitizer so as to target the photosensitizer to choroidal neovasculature.

REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 60/181,641, filed Feb. 10, 2000, the disclosure of which isincorporated herein by reference.

FIELD OF THE INVENTION

The invention relates generally to photodynamic therapy-based methodsand compositions for treating ocular conditions and, more specifically,the invention relates to photodynamic therapy-based methods andcompositions for treating ocular conditions characterized by unwantedchoroidal neovasculature.

BACKGROUND

Choroidal neovascularization can lead to hemorrhage and fibrosis, withresulting visual loss in a number of conditions of the eye, including,for example, age-related macular degeneration, ocular histoplasmosissyndrome, pathologic myopia, angioid streaks, idiopathic disorders,choroiditis, choroidal rupture, overlying choroid nevi, and certaininflammatory diseases. One of the disorders, namely, age-related maculardegeneration (AMD), is the leading cause of severe vision loss in peopleaged 65 and above (Bressler et al. (1988) SURV. OPHTHALMOL. 32, 375-413,Guyer et al. (1986) ARCH. OPHTHALMOL. 104, 702-705, Hyman et al. (1983)AM. J. EPIDEMIOL. 188, 816-824, Klein & Klein (1982) ARCH. OPHTHALMOL.100, 571-573, Leibowitz et al. (1980) SURV. OPHTHALMOL. 24, 335-610).Although clinicopathologic descriptions have been made, little isunderstood about the etiology and pathogenesis of the disease.

Dry AMD is the more common form of the disease, characterized by drusen,pigmentary and atrophic changes in the macula, with slowly progressiveloss of central vision. Wet or neovascular AMD is characterized bysubretinal hemorrhage, fibrosis and fluid secondary to the formation ofchoroidal neovasculature (CNV), and more rapid and pronounced loss ofvision. While less common than dry AMD, neovascular AMD accounts for 80%of the severe vision loss due to AMD. Approximately 200,000 cases ofneovascular AMD are diagnosed yearly in the United States alone.

Currently there is no treatment for dry AMD. Until recently, laserphotocoagulation has been the only therapy available for selected casesof neovascular AMD. Unfortunately, the majority of patients withneovascular AMD do not meet the criteria for laser photocoagulationtherapy. Approximately 85% of patients with neovascular AMD have poorlydefined, occult, or relatively extensive subfoveal choroidalneovascularization, none of which is amenable to laser therapy. Inaddition, laser photocoagulation relies on thermal damage to the CNVtissue, which damages the overlying neurosensory retina with consequentloss of visual function. Laser photocoagulation also is plagued byrecurrences that occur in approximately 50% of cases.

Photodynamic therapy (PDT) has shown promising results as a newtreatment for removing unwanted CNV and for treating neovascular AMD(Miller et al. (1999) ARCHIVES OF OPHTHALMOLOGY 117: 1161-1173,Schmidt-Erfurth et al. (1999) ARCHIVES OF OPHTHALMOLOGY 117: 1177-1187,TAP Study Group (1999) ARCHIVES OF OPHTHALMOLOGY 117: 1329-45, Husain etal. (1999) PHILADELPHIA: MOSBY; 297-307). PDT involves the systemicadministration of a photosensitizer or PDT dye (photosensitizer) thataccumulates in proliferating tissues such as tumors and newly formedblood vessels; followed by irradiation of the target tissue withlow-intensity, non-thermal light at a wavelength corresponding to theabsorption peak of the dye (Oleinick et al. (1998) RADIATION RESEARCH:150: S146-S156). Excitation of the dye leads to the formation of singletoxygen and free radicals—better known as reactive oxygen species whichcause photochemical damage to the target tissue (Weishaupt et al. (1976)CANCER RES. 36: 2326-2329).

Studies using PDT for the treatment of CNV have demonstrated that, withthe proper treatment parameters of photosensitizer dose, laser lightdose, and timing of irradiation, relative selective damage toexperimental CNV can be achieved, sparing retinal vessels, largechoroidal vessels, and with minimal changes in the neurosensory retina(Husain et al. (1996) ARCH OPHTALMOL. 114: 978-985, Husain et al. (1997)SEMINARS IN OPHTHALMOLOGY 12: 14-25, Miller et al. (1995) ARCHOPHTHALMOL. 113: 810-818, Kramer et al. (1996) OPHTHALMOLOGY 103(3):427-438). Moreover, a PDT-based procedure using a green porphyrin dyerecently has been approved in a variety of countries for use in thetreatment of neovascular AMD.

During clinical studies, however, it has been found that recurrence ofleakage appears in at least a portion of the CNV by one to three monthspost-treatment. Increasing photosensitizer or light doses do not appearto prevent this recurrence, and can even lead to undesired non-selectivedamage to retinal vessels (Miller et al. (1999) ARCHIVES OFOPHTHALMOLOGY 117: 1161-1173). Several multicenter Phase 3 trials areunderway to study repeated PDT treatments, applied every three months.The interim data look promising in terms of decreased rates of moderatevision loss (TAP Study Group (1999) ARCHIVES OF OPHTHALMOLOGY 117:1329-45). The necessity for repeated PDT treatments can nevertheless beexpected to lead to cumulative damage to the retinal pigment epithelium(RPE) and choriocapillaris, which may lead to progressivetreatment-related vision loss.

Therefore, there is still a need for improved PDT-based methods thatincrease the efficacy and selectivity of treatment, and which reduce ordelay a recurrence of the disorder.

SUMMARY OF THE INVENTION

The present invention is directed to PDT-based methods and compositionsfor treating ocular conditions associated with unwanted choroidalneovasculature. Such conditions include, for example, neovascular AMD,ocular histoplasmosis syndrome, pathologic myopia, angioid streaks,idiopathic disorders, choroiditis, choroidal rupture, overlying choroidnevi, and certain inflammatory diseases. The invention provides a moreeffective PDT-based method for treating unwanted CNV that has one ormore of the following advantages: increased efficacy of treatment;increased selectivity for CNV; and reduced or delayed recurrence of thecondition following PDT.

In one aspect, the invention provides a method of treating unwanted CNVin a mammal, wherein the CNV comprises endothelial cells, for example,capillary endothelial cells. The method comprises the steps of: (a)administering to the mammal, for example, a primate, preferably, ahuman, an anti-angiogenesis factor in an amount sufficient to permit aneffective amount to localize in the CNV; (b) administering to the mammalan amount of a photosensitizer (PDT dye) sufficient to permit aneffective amount to localize in the CNV; and (c) irradiating the CNVwith laser light such that the light is absorbed by the photosensitizerso as to occlude the CNV. During practice of this method, the damage toendothelial cells disposed within the choroidal neovasculature isgreater than the damage experienced by endothelial cells in a similartreatment lacking administration of the anti-angiogenesis factor.Furthermore, the anti-angiogenesis factor can potentiate thecytotoxicity of PDT. For example, the anti-angiogenesis factor and thePDT may act synergistically to selectively kill capillary endothelialcells, while at the same time sparing retinal cells, for example,retinal pigment epithelial cells and cells disposed in the neurosensoryretina, for example, photoreceptor cells and Mueller cells.

The anti-angiogenesis factor can enhance the selectivity of the PDT by,for example, occluding the CNV while at the same sparing surroundingblood vessels, for example, normal choroidal and retinal vasculature,and/or tissue, for example, the overlying neurosensory retina.Accordingly, inclusion of the anti-angiogenesis factor makes the PDTmethod more selective for capillary endothelial cells. Furthermore,practice of the invention can slow down or delay the recurrence ofchoroidal neovasculature.

A variety of anti-angiogenesis factors may be used in the invention.Useful anti-angiogenesis factors, include, for example: angiostatin;endostatin; a peptide containing a RGD tripeptide sequence and capableof binding the αvβ integrin; a COX-2 selective inhibitor; halofuginone;anecotave acetate; antibodies and other peptides that bind vascularendothelial growth factor receptor; antibodies, other peptides, andnucleic acids that bind vascular endothelial growth factor to prevent orreduce its binding to its cognate receptor; tyrosine kinase inhibitors;thrombospondin-1; anti-epidermal growth factor; hepatocyte growthfactor; thromboxane; and pigment endothelial-derived growth factor.Preferred anti-angiogenic factors include angiostatin, endostatin andpigment epithelium-derived growth factor.

The anti-angiogenesis factor may, under certain circumstances, beco-administered simultaneously with the photosensitizer. In a preferredembodiment, however, the anti-angiogenesis factor is administered to themammal prior to administration of the photosensitizer.

In another aspect, the invention provides a method of treating unwantedCNV in a mammal. The method comprises the steps of: (a) administering toa mammal, for example, a primate, preferably, a human, an amount of aphotosensitizer to permit an effective amount to localize in the CNV,the photosensitizer comprising a targeting moiety that bindspreferentially to cell surface ligands disposed on endothelial cells,for example, capillary endothelial cells, present in the CNV; and (b)irradiating the CNV with laser light such that the light is absorbed bythe photosensitizer so as to occlude the CNV. The targeting moietiesbind preferentially to CNV and, therefore, can increase the effectiveconcentration of photosensitizer in the CNV relative to surroundingcells and tissues. Accordingly, such a method increases the selectivityof the PDT method for CNV while sparing surrounding retinal and largechoroidal blood vessels and overlying neurosensory retina.

The targeting moiety can be any molecule, for example, a protein,peptide, nucleic acid, peptidyl-nucleic acid, organic molecule orinorganic molecule that has an affinity for endothelial cells withinCNV. However, targeting proteins and peptides are preferred. Forexample, the targeting peptide can be a peptide that targets αvβintegrin, for example, αvβ 3 integrin or αvβ 5 integrin. Alternatively,the targeting peptide can be an antibody, for example, a monoclonalantibody or an antigen binding fragment thereof, a polyclonal antibodyor an antigen binding fragment thereof, or a biosynthetic antibodybinding site that binds preferentially to a cell surface ligand disposedat elevated concentrations or densities in CNV. By way of example, thetargeting moiety may be an antibody that binds specifically to thevascular endothelial growth factor receptor.

In another aspect, the invention provides a method of treating unwantedCNV in a mammal. The method comprises the steps of: (a) administering tothe mammal, for example, a primate, and more preferably, a human, anapoptosis-modulating factor in an amount sufficient to permit aneffective amount to localize in the CNV or tissue surrounding the CNV;(b) administering to the mammal an amount of photosensitizer sufficientto permit an effective amount of localize in the CNV; and (c)irradiating the CNV with laser light such that the light is absorbed bythe photosensitizer so as to occlude the CNV. Cytotoxicity of the PDTcan be enhanced and/or made more specific for CNV relative to a similartreatment lacking the apoptosis-modulating factor.

The apoptosis-modulating factor may be any molecule, for example, aprotein, peptide, nucleic acid, peptidyl-nucleic acid, organic moleculeor inorganic molecule, that enhances or stimulates apoptosis in cells ortissues of the CNV or that represses apoptosis in cells or tissuessurrounding the CNV. In a preferred embodiment, the apoptosis-modulatingfactor is a peptide capable of inducing apoptosis in cells, for example,endothelial cells, present in CNV. The peptide may comprise, forexample, an amino sequence comprising, in an N- to C-terminal direction,KLAKLAKKLAKLAK (SEQ ID NO: 1) which is designed to be non-toxic outsidecells, but which is toxic when internalized into target cells because itdisrupts mitochondrial membranes. Furthermore, this peptide may betargeted towards endothelial cells by inclusion of a targeting aminoacid sequence, for example, in an N- to C-terminal direction, ACDCRGDCFC(SEQ ID NO: 2), also known as RGD-4C.

The apoptosis-modulating factor may be co-administered simultaneouslywith the photosensitizer. However, in a preferred embodiment, theapoptosis-modulating factor is administered to the primate beforeadministration of the photosensitizer and/or irradiation.

In all the foregoing methods, it is contemplated that anyphotosensitizer useful in PDT may be useful in the practice of theinvention. Preferred photosensitizers include, for example, amino acidderivatives, azo dyes, xanthene derivatives, chlorins, tetrapyrrolederivatives, phthalocyanines, and assorted other photosensitizers.However, preferred photosensitizers, include, for example, lutetiumtexaphyrin, benzoporphyrin and derivatives thereof, and hematoporphyrinand derivatives thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features, and advantages of the presentinvention, as well as the invention itself, may be more fully understoodfrom the following description of preferred embodiments, when readtogether with the accompanying drawings, in which:

FIGS. 1A and 1B are bar charts showing the in vitro survival of bovineretinal capillary endothelial (BRCE) cells (FIG. 1A) and retinal pigmentepithelial (RPE) cells (FIG. 1B) upon exposure to Lutetium Texaphyrin(Lu-Tex)/PDT in the presence or absence of angiostatin. Cells wereplated and exposed to angiostatin 18 hours before Lu-Tex/PDT. Thesurviving fraction was measured using a 1-week proliferation assay. Datarepresent the mean of triplicate experiments ±SD;

FIGS. 2A-2C are graphs showing the kinetics of Caspase 3-like activationfollowing Lu-Tex/PDT in BRCE (diamonds) and RPE (squares). BRCE and RPEcells were exposed to Lu-Tex/PDT at fluences of 10 J/cm² (FIG. 2A), 20J/cm² (FIG. 2B) and 40 J/cm² (FIG. 2C). At the indicated timesthereafter, cells were collected and lysed. Aliquots (50 μg of protein)were incubated with Ac-DEVD-AFC at 37° C. for 30 min. The amount offluorochrome released was determined by comparison to a standard curvein lysis buffer and the data represent the mean of three independentexperiments; and

FIG. 3 is a graph showing Caspase 3-like activity in BRCE followingAngiostatin/Lu-Tex/PDT versus Lu-Tex/PDT alone. BRCE were exposed toangiostatin (500 ng/ml) alone (diamonds), Lu-Tex/PDT (20 J/cm²(squares), 40 J/cm² (crosses)) alone and combination ofangiostatin/Lu-Tex/PDT (triangles). At the indicated times thereafter,cells were collected and lysed. Aliquots (50 μg of protein) wereincubated with Ac-DEVD-AFC at 37° C. for 30 min. The amount offluorochrome released was determined by comparison to a standard curvein lysis buffer and the data represent the means of three independentexperiments.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to an improved PDT-based method for treatingocular conditions characterized as having unwanted CNV. Such conditionsinclude, for example, neovascular AMD, ocular histoplasmosis syndrome,pathologic myopia, angioid streaks, idiopathic disorders, choroiditis,choroidal rupture, overlying choroid nevi, and certain inflammatorydiseases. The invention provides one or more of the followingadvantages: increased efficacy of treatment; increased selectivity forCNV; and reduced or delayed recurrence of the condition following PDT.

The method of the invention relates to a PDT-based method of treatingunwanted target CNV. The method requires administration of aphotosensitizer to a mammal in need of such treatment in an amountsufficient to permit an effective amount (i.e., an amount sufficient tofacilitate PDT) of the photosensitizer to localize in the target CNV.After administration of the photosensitizer, the CNV then is irradiatedwith laser light under conditions such that the light is absorbed by thephotosensitizer. The photosensitizer, when activated by the light,generates singlet oxygen and free radicals, for example, reactive oxygenspecies, that result in damage to surrounding tissue. For example,PDT-induced damage of endothelial cells results in platelet adhesion anddegranulation, leading to stasis and aggregration of blood cells andvascular occlusion.

An increase in efficacy and/or selectivity of the PDT, and/or reductionor delay of recurrence of the CNV can be achieved by (i) administeringan anti-angiogenic factor to the mammal prior to or concurrent withadministration of the photosensitizer, (ii) using a photosensitizer witha targeting molecule that targets the photosensitizer to the CNV, (iii)administering an apoptosis-modulating factor to the mammal prior to orconcurrent with administration of the photosensitizer, (iv) acombination of any two of the foregoing, for example, a combination ofthe anti-angiogenesis factor and the targeted photosensitizer, acombination of the anti-angiogenesis factor and the apoptosis modulatingagent, or a combination of the targeted photosenitizer and the apoptosismodulating agent, or (v) a combination of all three of the foregoing.

It is contemplated that a variety of photosensitizers useful in PDT maybe useful in the practice of the invention and include, for example,amino acid derivatives, azo dyes, xanthene derivatives, chlorins,tetrapyrrole derivatives, phthalocyanines, and assorted otherphotosensitizers.

Amino acid derivatives include, for example, 5-aminolevulinic acid (Berget al. (1997) PHOTOCHEM. PHOTOBIOL 65: 403-409; El-Far et al. (1985)CELL. BIOCHEM. FUNCTION 3, 115-119). Azo dyes, include, for example,Sudan I, Sudan II, Sudan III, Sudan IV, Sudan Black, Disperse Orange,Disperse Red, Oil Red O, Trypan Blue, Congo Red, β-carotene (Mosky etal. (1984) Exp. RES. 155, 389-396). Xanthene derivatives, include, forexample, rose bengal.

Chlorins include, for example, lysyl chlorin p6 (Berg et al. (1997)supra) and etiobenzochlorin (Berg et al. (1997) supra), 5, 10, 15,20-tetra (m-hydroxyphenyl) chlorin (M-THPC), N-aspartyl chlorin e6(Dougherty et al. (1998) J. NATL. CANCER INST. 90: 889-905), andbacteriochlorin (Korbelik et al. (1992) J. PHOTOCHEM. PHOTOBIOL. 12:107-119).

Tetrapyrrole derivatives include, for example, lutetium texaphrin(Lu-Tex, PCI-0123) (Dougherty et al. (1998) supra, Young et al. (1996)PHOTOCHEM. PHOTOBIOL. 63: 892-897); benzoporphyrin derivative (BPD)(U.S. Pat. Nos. 5,171,749, 5,214,036, 5,283,255, and 5,798,349, Jori etal. (1990) LASERS MED. SCI. 5, 115-120), benzoporphyrin derivative monoacid (BPD-MA) (U.S. Pat. Nos. 5,171,749, 5,214,036, 5,283,255, and5,798,349, Berg et al. (1997) supra, Dougherty et al. (1998) supra),hematoporphyrin (Hp) (Jori et al. (1990) supra), hematoporphyrinderivatives (HpD) (Berg et al. (1997) supra, West et al. (1990) IN. J.RADIAT. BIOL. 58: 145-156), porfimer sodium or Photofrin (PHP) (Berg etal. (1997) supra), Photofrin II (PII) (He et al. (1994) PHOTOCHEM.PHOTOBIOL. 59: 468-473), protoporphyrin IX (PpIX) (Dougherty et al.(1998) supra, He et al. (1994) supra), meso-tetra (4-carboxyphenyl)porphine (TCPP) (Musser et al. (1982) RES. COMMUN. CHEM. PATHOL.PHARMACOL. 2, 251-259), meso-tetra (4-sulfonatophenyl) porphine (TSPP)(Musser et al. (1982) supra), uroporphyrin I (UROP-I) (El-Far et al.(1985) CELL. BIOCHEM. FUNCTION 3, 115-119), uroporphyrin III (UROP-III)(El-Far et al. (1985) supra), tin ethyl etiopurpurin (SnET2), (Doughertyet al. (1998) supra 90: 889-905) and 13, 17-bis[1-carboxypropionyl]carbamoylethyl-8-etheny-2-hydroxy-3-hydroxyiminoethylidene-2,7,12,18-tetranethyl 6 porphyrin sodium (ATX-S10(Na)) Mori et al.(2000) JPN. J. CANCER RES. 91:753-759, Obana et al. (2000) ARCH.OPHTHALMOL. 118:650-658, Obana et al. (1999) LASERS SURG. MED.24:209-222).

Phthalocyanines include, for example, chloroaluminum phthalocyanine(AlPcCl) (Rerko et al. (1992) PHOTOCHEM. PHOTOBIOL. 55, 75-80), aluminumphthalocyanine with 2-4 sulfonate groups (AlPcS₂₋₄) (Berg et al. (1997)supra, Glassberg et al. (1991) LASERS SURG. MED. 11, 432-439),chloro-aluminum sulfonated phthalocyanine (CASPc) (Roberts et al. (1991)J. NATL. CANCER INST. 83, 18-32), phthalocyanine (PC) (Jori et al.(1990) supra), silicon phthalocyanine (Pc4) (He et al. (1998) PHOTOCHEM.PHOTOBIOL. 67: 720-728, Jori et al. (1990) supra), magnesiumphthalocyanine (Mg²⁺-PC) (Jori et al. (1990) supra), zinc phthalocyanine(ZnPC) (Berg et al. (1997) supra). Other photosensitizers include, forexample, thionin, toluidine blue, neutral red and azure c.

However, preferred photosensitizers, include, for example, LutetiumTexaphyrin (Lu-Tex), a new generation photosensitizer currently inclinical trial for CNV because of its favorable clinical propertiesincluding absorption at about 730 nm permitting deep tissue penetrationand rapid clearance which is available from Alcon Laboratories, FortWorth, Tex. Other preferred photosensitizers, include benzoporhyrin andbenzoporphyrin derivatives, for example, BPD-MA and BPD-DA, availablefrom QLT Phototherapeutics, Inc., Vancouver, Canada.

The photosensitizer preferably is formulated into a delivery system thatdelivers high concentrations of the photosensitizer to the CNV. Suchformulations may include, for example, the combination of aphotosensitizer with a carrier that delivers higher concentrations ofthe photosensitizer to CNV and/or coupling the photosensitizer to aspecific binding ligand that binds preferentially to a specific cellsurface component of the CNV.

In one preferred embodiment, the photosensitizer can be combined with alipid based carrier. For example, liposomal formulations have been foundto be particularly effective at delivering the photosensitizer, greenporphyrin, and more particularly BPD-MA to the low-density lipoproteincomponent of plasma, which in turn acts as a carrier to deliver thephotosensitizer more effectively to the CNV. Increased numbers of LDLreceptors have been shown to be associated with CNV, and by increasingthe partitioning of the photosenstizer into the lipoprotein phase of theblood, it may be delivered more efficiently to the CNV. Certainphotosensitizers, for example, green porphyrins, and in particularBPD-MA, interact strongly with lipoproteins. LDL itself can be used as acarrier, but LDL is considerably more expensive and less practical thana liposomal formulation. LDL, or preferably liposomes, are thuspreferred carriers for the green porphyrins since green porphyrinsstrongly interact with lipoproteins and are easily packaged inliposomes. Compositions of green porphyrins formulated as lipocomplexes,including liposomes, are described, for example, in U.S. Pat. Nos.5,214,036, 5,707,608 and 5,798,349. Liposomal formulations of greenporphyrin can be obtained from QLT Phototherapeutics, Inc., Vancouver,Canada. It is contemplated that certain other photosensitizers maylikewise be formulated with lipid carriers, for example, liposomes orLDL, to deliver the photosensitizer to CNV.

Furthermore, the photosensitizer can be coupled to a specific bindingligand that binds preferentially to a cell surface component of the CNV,for example, neovascular endothelial homing motif. It appears that avariety of cell surface ligands are expressed at higher levels in newblood vessels relative to other cells or tissues.

Endothelial cells in new blood vessels express several proteins that areabsent or barely detectable in established blood vessels (Folkman (1995)NATURE MEDICINE 1:27-31), and include integrins (Brooks et al. (1994)SCIENCE 264: 569-571; Friedlander et al. (1995) SCIENCE 270: 1500-1502)and receptors for certain angiogenic factors like vascular endothelialgrowth factor (VEGF). In vivo selection of phage peptide libraries havealso identified peptides expressed by the vasculature that areorgan-specific, implying that many tissues have vascular “addresses”(Pasqualini et al. (1996) NATURE 380: 364-366). It is contemplated thata suitable targeting moiety can direct a photosensitizer to the CNVendothelium thereby increasing the efficacy and lowering the toxicity ofPDT.

Several targeting molecules may be used to target photosensitizers tothe neovascular endothelium. For example, α-v integrins, in particularα-v β3 and α-v β5, appear to be expressed in ocular neovascular tissue,in both clinical specimens and experimental models (Corjay et al. (1997)INVEST. OPHTHALMOL. VIS. SCI. 38, S965; Friedlander et al. (1995)supra). Accordingly, molecules that preferentially bind α-v integrinscan be used to target the photosensitizer to CNV. For example, cyclicpeptide antagonists of these integrins have been used to inhibitneovascularization in experimental models (Friedlander et al. (1996)PROC. NATL. ACAD. SCI. USA 93:9764-9769). A peptide motif having anamino acid sequence, in an N- to C-terminal direction, ACDCRGDCFC (SEQID NO: 2)—also know as RGD-4C—has been identified that selectively bindsto human α-v integrins and accumulates in tumor neovasculature moreeffectively than other angiogenesis targeting peptides (Arap et al.(1998) NATURE 279:377-380; Ellerby et al. (1999) NATURE MEDICINE 5:1032-1038). Angiostatin may also be used as a targeting molecule for thephotosensitizer. Studies have shown, for example, that angiostatin bindsspecifically to ATP synthase disposed on the surface of humanendothelial cells (Moser et al. (1999) PROC. NATL. ACAD. SCI. USA96:2811-2816)

Another potential targeting molecule is an antibody for vascularendothelial growth factor receptor (VEGF-2R). Clinical and experimentalevidence strongly supports a role for VEGF in ocular neovascularization,particularly ischemia-associated neovascularization (Adamis et al.(1996) ARCH. OPHTHALMOL. 114:66-71; Tolentino et al. (1996) ARCH.OPHTHALMOL. 114:964-970; Tolentino et al. (1996) OPHTHALMOLOGY103:1820-1828). Antibodies to the VEGF receptor (VEGFR-2 also known asKDR) may also bind preferentially to neovascular endothelium. As usedherein, the term “antibody” includes, for example, a monoclonal antibodyor an antigen binding fragment thereof (for example, an Fv, Fab, Fab′ oran (Fab′)₂ molecule), a polyclonal antibody or an antigen bindingfragment thereof, or a biosynthetic antibody binding site, for example,an sFv (U.S. Pat. Nos. 5,091,513; 5,132,405; 5,258,498; and 5,482,858)that binds specifically to a target ligand. As used herein, the termsbinds “specifically” or “preferentially” are understood to mean that thetargeting molecule, for example, the antibody, binds to thecomplementary or target ligand with a binding affinity of at least 10⁵,and more preferably 10⁷ M⁻¹.

The targeting molecule may be synthesized using methodologies known andused in the art. For example, proteins and peptides may be synthesizedusing conventional synthetic peptide chemistries or expressed asrecombinant proteins or peptides in a recombinant expression system(see, for example, “Molecular Cloning” Sambrook et al. eds, Cold SpringHarbor Laboratories). Similarly, antibodies may be prepared and purifiedusing conventional methodologies, for example, as described in“Practical Immunology”, Butt, W. R. ed., 1984 Marcel Deckker, New Yorkand “Antibodies, A Laboratory Approach” Harlow et al., eds. (1988), ColdSpring Harbor Press. Once created, the targeting agent may be coupled tothe photosensitizer using standard coupling chemistries, using, forexample, conventional cross linking reagents, for example,heterobifunctional cross linking reagents available, for example, fromPierce, Rockford, Ill.

Once formulated, the photosensitizer may be administered in any of awide variety of ways, for example, orally, parenterally, or rectally.Parenteral administration, such as intravenous, intramuscular, orsubcutaneous, is preferred. Intravenous injection is especiallypreferred. The dose of photosensitizer can vary widely depending on thetissue to be treated; the physical delivery system in which it iscarried, such as in the form of liposomes; or whether it is coupled to atarget-specific ligand, such as an antibody or an immunologically activefragment.

It should be noted that the various parameters used for effective,selective photodynamic therapy in the invention are interrelated.Therefore, the dose should also be adjusted with respect to otherparameters, for example, fluence, irradiance, duration of the light usedin PDT, and time interval between administration of the dose and thetherapeutic irradiation. All of these parameters should be adjusted toproduce significant damage to CNV without significant damage to thesurrounding tissue.

Typically, the dose of photosensitizer used is within the range of fromabout 0.1 to about 20 mg/kg, preferably from about 0.15 to about 5.0mg/kg, and even more preferably from about 0.25 to about 2.0 mg/kg.Furthermore, as the dosage of photosensitizer is reduced, for example,from about 2 to about 1 mg/kg in the case of green porphyrin or BPD-MA,the fluence required to close CNV may increase, for example, from about50 to about 100 Joules/cm². Similar trends may be observed with theother photosensitizers discussed herein.

After the photosensitizer has been administered, the CNV is irradiatedat a wavelength typically around the maximum absorbance of thephotosensitizer, usually in the range from about 550 nm to about 750 nm.A wavelength in this range is especially preferred for enhancedpenetration into bodily tissues. Preferred wavelengths used for certainphotosensitizers include, for example, about 690 nm for benzoporphyrinderivative mono acid, about 630 nm for hematoporphyrin derivative, about675 nm for chloro-aluminum sulfonated phthalocyanine, about 660 nm fortin ethyl etiopurpurin, about 730 nm for lutetium texaphyrin, about 670nm for ATX-S10(NA), about 665 nm for N-aspartyl chlorin e6, and about650 nm for 5, 10, 15, 20-tetra (m-hydroxyphenyl) chlorin.

As a result of being irradiated, the photosensitizer in its tripletstate is thought to interact with oxygen and other compounds to formreactive intermediates, such as singlet oxygen and reactive oxygenspecies, which can disrupt cellular structures. Possible cellulartargets include the cell membrane, mitochondria, lysosomal membranes,and the nucleus. Evidence from tumor and neovascular models indicatesthat occlusion of the vasculature is a major mechanism of photodynamictherapy, which occurs by damage to the endothelial cells, withsubsequent platelet adhesion, degranulation, and thrombus formation.

The fluence during the irradiating treatment can vary widely, dependingon the type of photosensitizer used, the type of tissue, the depth oftarget tissue, and the amount of overlying fluid or blood. Fluencespreferably vary from about 10 to about 400 Joules/cm² and morepreferably vary from about 50 to about 200 Joules/cm². The irradiancevaries typically from about 50 mW/cm² to about 1800 mW/cm², morepreferably from about 100 mW/cm² to about 900 mW/cm², and mostpreferably in the range from about 150 mW/cm² to about 600 mW/cm². It iscontemplated that for many practical applications, the irradiance willbe within the range of about 300 mW/cm² to about 900 mW/cm². However,the use of higher irradiances may be selected as effective and havingthe advantage of shortening treatment times.

The time of light irradiation after administration of thephotosensitizer may be important as one way of maximizing theselectivity of the treatment, thus minimizing damage to structures otherthan the target tissues. The optimum time following photosensitizeradministration until light treatment can vary widely depending on themode of administration, the form of administration such as in the formof liposomes or as a complex with LDL, and the type of target tissue.For example, benzoporphyrin derivative typically becomes present withinthe target neovasculature within one minute post administration andpersists for about fifty minutes, lutetium texaphyrin typically becomespresent within the target neovasculature within one minute postadministration and persists for about twenty minutes, N-aspartyl chlorine6 typically becomes present within the target neovasculature within oneminute post administration and persists for about twenty minutes, androse bengal typically becomes present in the target vasculature withinone minute post administration and persists for about ten minutes.

Effective vascular closure generally occurs at times in the range ofabout one minute to about three hours following administration of thephotosensitizer. However, as with green porphyrins, it is undesirable toperform the PDT within the first five minutes following administrationto prevent undue damage to retinal vessels still containing relativelyhigh concentrations of photosensitizer.

The efficacy of PDT may be monitored using conventional methodologies,for example, via fundus photography or angiography. Closure can usuallybe observed angiographically by hypofluorescence in the treated areas inthe early angiographic frames. During the later angiographic frames, acorona of hyperfluorescence may begin to appear which then fills thetreated area, possibly representing leakage from the adjacentchoriocapillaris through damaged retinal pigment epithelium in thetreated area. Large retinal vessels in the treated area typicallyperfuse following photodynamic therapy.

Minimal retinal damage is generally found on histopathologic correlationand is dependent on the fluence and the time interval after irradiationthat the photosensitizer is administered. It is contemplated that thechoice of appropriate photosensitizer, dosage, mode of administration,formulation, timing post administration prior to irradiation, andirradiation parameters may be determined empirically.

It is contemplated that a variety of anti-angiogenic factors may becombined with PDT to treat unwanted CNV. The anti-angiogenesis factorcan potentiate the cytotoxity of the PDT thereby enhancing occlusion ofthe choroidal neovasculature. In addition, the anti-angiogenesis factorcan enhance the selectivity of PDT, for example, by occluding the CNVwhile at the same sparing the surrounding blood vessels, for example,the retinal and large choroidal blood vessels and/or surrounding tissue,for example, the retinal epithelium. Furthermore, the anti-angiogenesisfactor can be used to reduce or delay the recurrence of the condition.

The term “anti-angiogensis factor” is understood to mean any molecule,for example, a protein, peptide, nucleic acid (ribose nucleic acid (RNA)or deoxyribose nucleic acid (DNA)), peptidyl nucleic acid, organiccompound or inorganic compound, that reduces or inhibits the formationof new blood vessels in a mammal. It is contemplated that usefulangiogenesis inhibitors, if not already known, may be identified using avariety of assays well known and used in the art. Such assays include,for example, the bovine capillary endothelial cell proliferation assay,the chick chorioallantoic membrane (CAM) assay or the mouse cornealassay. However, the CAM assay is preferred (see, for example, O'Reillyet al. (1994) CELL 79: 315-328 and O'Reilly et al. (1997) CELL 88:277-285). Briefly, embryos with intact yolks are removed from fertilizedthree day old white eggs and placed in a petri dish. After incubation at37° C., 3% CO₂ for three days, a methylcellulose disk containing theputative angiogenesis inhibitor is applied to the chorioallantoicmembrane of an individual embryo. After incubation for about 48 hours,the chorioallantoic membranes are observed under a microscope forevidence of zones of inhibition.

Numerous anti-angiogenesis factors are well known and thoroughlydocumented in the art (see, for example, PCT/US99/08335). Examples ofanti-angiogenesis factors useful in the practice of the invention,include, for example, protein/peptide inhibitors of angiogenesis suchas: angiostatin, a proteolytic fragment of plasminogen (O'Reilly et al.(1994) CELL 79: 315-328, and U.S. Pat. Nos. 5,733,876; 5,837,682; and5,885,795) including full length amino acid sequences of angiostatin,bioactive fragments thereof, and analogs thereof; endostatin, aproteolytic fragment of collagen XVIII (O'Reilly et al. (1997) CELL 88:277-285, Cirri et al. (1999) INT. BIOL. MARKER 14: 263-267, and U.S.Pat. No. 5,854,205) including full length amino acid sequences ofendostatin, bioactive fragments thereof, and analogs thereof; peptidescontaining the RGD tripeptide sequence and capable of binding theα_(v)β₃ integrin (Brooks et al. (1994) CELL 79: 1157-1164, Brooks et al.(1994) SCIENCE 264: 569-571); certain antibodies and antigen bindingfragments thereof and peptides that bind preferentially to the α_(v)β₃integrin found on tumor vascular epithelial cells (Brooks et al., supra,Friedlander et al. (1996) PROC. NATL. ACAD. SCI. USA 93: 9764-9769);certain antibodies and antigen binding fragments thereof and peptidesthat bind preferentially to the epidermal growth factor receptor(Ciardello et al. (1996) J. NATL. CANCER INST. 88: 1770-1776, Ciardelloet al. (2000) CLIN. CANCER RES. 6:3739-3747); antibodies, proteins,peptides and/or nucleic acids that bind preferentially to and neutralizevascular endothelial growth factor (Adarnis et al. (1996) ARCH OPTHALMOL114:66-71), antibodies, proteins, and/or peptides that bindpreferentially to and neutralize vascular endothelial growth factorreceptor; anti-fibroblast growth factor, anti-epidermal growth factor(Ciardiello et al. (2000) CLIN. CANCER RES. 6: 3739-3747) including fulllength amino acid sequences, bioactive fragments and analogs thereof,and pigment epithelium-derived growth factor (Dawson (1999) SCIENCE2035: 245-248) including full length amino acid sequences, bioactivefragments and analogs thereof. Bioactive fragments refer to portions ofthe intact protein that have at least 30%, more preferably at least 70%,and most preferably at least 90% of the biological activity of theintact proteins. Analogs refer to species and allelic variants of theintact protein, or amino acid replacements, insertions or deletionsthereof that have at least 30%, more preferably at least 70%, and mostpreferably 90% of the biological activity of the intact protein.

Other angiogenesis inhibitors include, for example: COX-2 selectiveinhibitors (Masferrer et al. (1998) PROC. AMER. Assoc. CANCER RES. 39:271; Ershov et al. (1999) J. NEUROSCI. RES. 15: 254-261; Masferrer etal. (2000) CURR. MED. CHEM. 7: 1163-1170); tyrosine kinase inhibitors,for example, PD 173074 (Dimitroff et al. (1999) INVEST. NEW DRUGS 17:121-135), halofuginone (Abramovitch et al. (1999) NEOPLASIA 1: 321-329;Elkin et al. (1999) CANCER RES. 5: 1982-1988), AGM-1470 (Brem et al.(1993) J. PED. SURGERY 28: 1253-1257), angiogenic steroids, for example,hydrocortisone and anecortave acetate (Penn et al. (2000) INVEST.OPHTHALMOL. VIS. SCI. 42: 283-290), thrombospondin-1 (Shafiee et al.(2000) INVEST. OPHTHALMOL. VIS. SCI. 8: 2378-2388; Nor et al. (2000) J.VASC. RES. 37: 09-218), UCN-01 (Kruger et al. (1998-1999) INVASIONMETASTASIS 18: 209-218), CM101 (Sundell et al. (1997) CLIN. CANCER RES.3: 365-372); fumagillin and analogues such as AGM-1470 (Ingber et al.(1990) NATURE 348: 555-557), and other small molecules such asthalidomide (D'Amato et al. (1994) PROC. NATL. ACAD. SCI. USA 91:4082-4085).

Several cytokines including bioactive fragments thereof and analogsthereof have also been reported to have anti-angiogenic activity andthus can be useful in the practice of the invention. Examples include,for example, IL-12, which reportedly works through an IFN-γ-dependentmechanism (Voest et al. (1995) J. NATL. CANC. INST. 87: 581-586); IFN-α,which has been shown to be anti-angiogenic alone or in combination withother inhibitors (Brem et al. (1993) J. PEDIATR. SURG. 28: 1253-1257).Furthermore, the interferons IFN-α, IFN-β and IFN-γ reportedly haveimmunological effects, as well as anti-angiogenic properties, that areindependent of their anti-viral activities. However, preferredanti-angiogenic factors include endostatin and angiostatin.

The anti-angiogenesis factor may be synthesized using methodologiesknown and used in the art. For example, proteins and peptides may besynthesized and purified using conventional synthetic peptidechemistries and purification protocols, or expressed as recombinantproteins or peptides in a recombinant expression system (see, forexample, “Molecular Cloning” Sambrook et al. eds, Cold Spring HarborLaboratories). Similarly, antibodies may be prepared and purified usingconventional methodologies, for example, as described in “PracticalImmunology”, Butt, W. R. ed., 1984 Marcel Deckker, New York and“Antibodies, A Laboratory Approach” Harlow et al., eds. (1988), ColdSpring Harbor Press.

To the extent that the anti-angiogenesis factor is a nucleic acid orpeptidyl nucleic acid, such compounds may be synthesized by any of theknown chemical oligonucleotide and peptidyl nucleic acid synthesismethodologies known in the art (see, for example, PCT/EP92/20702 andPCT/US94/013523) and used in antisense therapy. Anti-senseoligonucleotide and peptidyl nucleic acid sequences, usually 10 to 100and more preferably 15 to 50 units in length, are capable of hybridizingto a gene and/or mRNA transcript and, therefore, may be used to inhibittranscription and/or translation of a target protein. It is appreciated,however, that oligoribonucleotide sequences generally are moresusceptible to enzymatic attack by ribonucleases than aredeoxyribonucleotide sequences. Hence, oligodeoxyribonucleotides arepreferred over oligoribonucleotides for in vivo use. In the case ofnucleotide sequences, phosphodiester linkages may be replaced bythioester linkages making the resulting molecules more resistant tonuclease degradation. Furthermore, it is appreciated that the peptidylnucleic acid sequences, unlike regular nucleic acid sequences, are notsusceptible to nuclease degradation and, therefore, are likely to havegreater longevity in vivo. Furthermore, it has been found that peptidylnucleic acid sequences bind complementary single stranded DNA and RNAstrands more strongly than corresponding DNA sequences (PCT/EP92/20702).Furthermore, to the extent that the anti-angiogenesis factor is anorganic or inorganic compound, such compounds may be synthesized,extracted and/or purified by standard procedures known in the art.

The type and amount of anti-angiogenesis factor to be administered maydepend upon the PDT and cell type to be treated. It is contemplated,however, that optimal anti-angiogenesis factors, modes of administrationand dosages may be determined empirically. The anti-angiogensis factormay be administered in a pharmaceutically acceptable carrier or vehicleso that administration does not otherwise adversely affect therecipient's electrolyte and/or volume balance. The carrier may comprise,for example, physiologic saline.

Protein, peptide or nucleic acid based angiogenesis inhibitors can beadministered at doses ranging, for example, from about 0.001 to about500 mg/kg, more preferably from about 0.01 to about 250 mg/kg, and mostpreferably from about 0.1 to about 100 mg/kg. For example, antibodiesthat bind vascular epithelial growth factor may be administeredintravenously at doses ranging from about 0.1 to about 5 mg/kg onceevery two to four weeks. Endostatin, for example, may be administeredintravenously on a daily basis at dosages ranging from about 1 to about50 mg/kg per day. With regard to intravitreal administration, theanti-angiogenesis factor, for example, antibodies that bind vascularepithelial growth factor, typically is administered periodically asboluses at dosages ranging from about 10 μg to about 5 mg/eye and morepreferably from about 100 μg to about 2 mg/eye.

The anti-angiogenesis factor preferably is administered to the mammalprior to PDT. Accordingly, it is preferable to administer theanti-angiogenesis factor prior to administration of the photosensitizer.The anti-angiogenesis factor, like the photosensitizer, may beadministered in any one of a wide variety of ways, for example, orally,parenterally, or rectally. However, parenteral administration, such asintravenous, intramuscular, subcutaneous, and intravitreal, ispreferred. Administration may be provided as a periodic bolus (forexample, intravenously or intavitreally) or as continuous infusion froman internal reservoir (for example, from a bioerodable implant disposedat an intra- or extra-ocular location) or from an external reservoir(for example, from an intravenous bag). The anti-angiogenesis factor maybe administered locally, for example, by continuous release from asustained release drug delivery device immobilized to an inner wall ofthe eye or via targeted trans-scleral controlled release into thechoroid (see, PCT/US00/00207).

The present invention, therefore, includes the use of ananti-angiogenesis factor in the preparation of a medicament fortreating, preferably by a PDT-based method, an ocular condition, thatpreferably is associated with choriodal neovasculature. Theanti-angiogenesis factor may be provided in a kit which optionally maycomprise a package insert with instructions for how to treat such acondition. A composition comprising both a photosensitizer and ananti-angiogenesis factor may be provided for use in the presentinvention. The composition may comprise a pharmaceutically acceptablecarrier or excipient. Thus, the present invention includes apharmaceutically acceptable composition comprising a photosensitizer andan anti-angiogenesis factor; as well as the composition for use inmedicine. More preferably, however, the invention is for use incombination therapy, whereby an anti-angiogenesis factor and aphotosensitizer are administered separately. Preferably theanti-angiogenesis factor is administered prior to administration of thephotosensitizer. Instructions for such administration may be providedwith the anti-angiogenesis factor and/or with the photosensitizer. Ifdesired, the anti-angiogenesis factor and photosensitizer may beprovided together in a kit, optionally including a package insert withinstructions for use. The anti-angiogenesis factor and photosensitizerpreferably are provided in separate containers. For each administration,the anti-angiogenesis factor and/or photosensitizer may be provided inunit-dosage or multiple-dosage form. Preferred dosages ofphotosensitizer and anti-angiogenic factor, however, are as describedabove.

In addition, the efficacy and selectivity of the PDT method may beenhanced by combining the PDT with an apoptosis-modulating factor. Anapoptosis-modulating factor can be any factor, for example, a protein(for example a growth factor or antibody), peptide, nucleic acid (forexample, an antisense oligonucleotide), peptidyl nucleic acid (forexample, an antisense molecule), organic molecule or inorganic molecule,that induces or represses apoptosis in a particular cell type. Forexample, it may be advantageous to prime the apoptotic machinery of CNVendothelial cells with an inducer of apoptosis prior to PDT so as toincrease their sensitivity to PDT. Endothelial cells primed in thismanner are contemplated to be more susceptible to PDT. This approach mayalso reduce the light dose (fluence) required to achieve CNV closure andthereby decreasing the level of damage on surrounding cells such as RPE.Alternatively, the cells outside the CNV may be primed with an arepressor of apoptosis so as to decrease their sensitivity to PDT. Inthis approach, the PDT at a particular fluence can become more selectivefor CNV.

Apoptosis involves the activation of a genetically determined cellsuicide program that results in a morphologically distinct form of celldeath characterized by cell shrinkage, nuclear condensation, DNAfragmentation, membrane reorganization and blebbing (Kerr et al. (1972)BR. J. CANCER 26: 239-257). At the core of this process lies a conservedset of proenzymes, called caspases, and two important members of thisfamily are caspases 3 and 7 (Nicholson et al. (1997) TIBS 22:299-306).Monitoring their activity can be used to assess on-going apoptosis.

It has been suggested that apoptosis is associated with the generationof reactive oxygen species, and that the product of the Bcl-₂ geneprotects cells against apoptosis by inhibiting the generation or theaction of the reactive oxygen species (Hockenbery et al. (1993) CELL 75:241-251, Kane et al. (1993) SCIENCE 262: 1274-1277, Veis et al. (1993)CELL 75: 229-240, Virgili et al. (1998) FREE RADICALS BIOL. MED. 24:93-101). Bcl-₂ belongs to a growing family of apoptosis regulatory geneproducts, which may either be death antagonists (Bcl-₂, BCl-x_(L).) ordeath agonists (Bax, Bak.) (Kroemer et al. (1997) NAT. MED. 3: 614-620).Control of cell death appears to be regulated by these interactions andby constitutive activities of the various family members (Hockenbery etal. (1993) CELL 75: 241-251). Several apoptotic pathways may coexist inmammalian cells that are preferentially activated in a stimulus-,stage-, context-specific and cell-type manner (Hakem et al. (1998) CELL94: 339-352).

The apoptosis-inducing factor preferably is a protein or peptide capableof inducing apoptosis in cells, for example, endothelial cells, disposedin the CNV. One apoptosis inducing peptide comprises an amino sequencehaving, in an N- to C-terminal direction, KLAKLAKKLAKLAK (SEQ ID NO: 1).This peptide reportedly is non-toxic outside cells, but become toxicwhen internalized into targeted cells by disrupting mitochondrialmembranes (Ellerby et al. (1999) supra). This sequence may be coupled,either by means of a crosslinking agent or a peptide bond, to atargeting domain, for example, the amino acid sequence known as ROD-4C(Ellerby et al. (1999) supra) that reportedly can direct theapoptosis-inducing peptide to endothelial cells. Otherapoptosis-inducing factors include, for example, constatin (Kamphaus etal. (2000) J. BIOL. CHEM. 14: 1209-1215), tissue necrosis factor α(Lucas et al. (1998) BLOOD 92: 4730-4741) including bioactive fragmentsand analogs thereof, cycloheximide (O'Connor et al. (2000) AM. J.PATHOL. 156: 393-398), tunicamycin (Martinez et al. (2000) ADV. Exp.MED. BIOL. 476: 197-208), adenosine (Harrington et al. (2000) AM. J.PHYSIOL. LUNG CELL MOL. PHYSIOL. 279: 733-742). Furthermore, otherapoptosis-inducing factors may include, for example, anti-sense nucleicacid or peptidyl nucleic acid sequences that reduce or turn off theexpression of one or more of the death antagonists, for example (Bcl-₂,Bcl-x_(L)). Antisense nucleotides directed against Bcl-₂ have been shownto reduce the expression of Bcl-₂ protein in certain lines together withincreased phototoxicity and susceptibility to apoptosis during PDT(Zhang et al. (1999) PHOTOCHEM PHOTOBIOL 69: 582-586). Furthermore, an18mer phosphorothiate oligonucleotide complementary to the first sixcodons of the Bcl-₂ open reading frame, and known as G3139, is beingtested in humans as a treatment for non-Hodgkins' lymphoma.

Apoptosis-repressing factors include, survivin including bioactivefragments and analogs thereof (Papapetropoulos et al. (2000) J. BIOL.CHEM. 275: 9102-9105), CD39 (Goepfert et al. (2000) MOL. MED. 6:591-603), BDNF (Caffe et al. (2001) INVEST. OPHTHALMOL. VIS. SCI. 42:275-82), FGF2 (Bryckaert et al. (1999) ONCOGENE 18: 7584-7593), Caspaseinhibitors (Ekert et al. (1999) CELL DEATH DIFFER 6: 1081-1068) andpigment epithelium-derived growth factor including bioactive fragmentsand analogs thereof. Furthermore, other apoptosis-repressing factors mayinclude, for example, anti-sense nucleic acid or peptidyl nucleic acidsequences that reduce or turn off the expression of one or more of thedeath agonists, for example (Bax, Bak).

To the extent that the apoptosis-modulating factor is a protein orpeptide, nucleic acid, peptidyl nucleic acid, organic or inorganiccompound, it may be synthesized and purified by one or more themethodologies described relating to the synthesis of theanti-angiogenesis factor.

The type and amount of apoptosis-modulating factor to be administeredmay depend upon the PDT and cell type to be treated. It is contemplated,however, that optimal apoptosis-modulating factors, modes ofadministration and dosages may be determined empirically. The apoptosismodulating factor may be administered in a pharmaceutically acceptablecarrier or vehicle so that administration does not otherwise adverselyaffect the recipient's electrolyte and/or volume balance. The carriermay comprise, for example, physiologic saline.

Protein, peptide or nucleic acid based apoptosis modulators can beadministered at doses ranging, for example, from about 0.001 to about500 mg/kg, more preferably from about 0.01 to about 250 mg/kg, and mostpreferably from about 0.1 to about 100 mg/kg. For example, nucleicacid-based apopotosis inducers, for example, G318, may be administeredat doses ranging from about 1 to about 20 mg/kg daily. Furthermore,antibodies may be administered intravenously at doses ranging from about0.1 to about 5 mg/kg once every two to four weeks. With regard tointravitreal administration, the apoptosis modulators, for example,antibodies, may be administered periodically as boluses a dosagesranging from about 10 μg to about 5 mg/eye and more preferably fromabout 100 μg to about 2 mg/eye.

The apoptosis-modulating factor preferably is administered to the mammalprior to PDT. Accordingly, it is preferable to administer theapoptosis-modulating factor prior to administration of thephotosensitizer. The apoptosis-modulating factor, like thephotosensitizer and anti-angiogenesis factor, may be administered in anyone of a wide variety of ways, for example, orally, parenterally, orrectally. However, parenteral administration, such as intravenous,intramuscular, subcutaneous, and intravitreal is preferred.Administration may be provided as a periodic bolus (for example,intravenously or intravitreally) or by continuous infusion from aninternal reservoir (for example, bioerodable implant disposed at anintra- or extra-ocular location) or an external reservoir (for example,and intravenous bag). The apoptosis modulating factor may beadministered locally, for example, by continuous release from asustained release drug delivery device immobilized to an inner wall ofthe eye or via targeted trans-scleral controlled release into thechoroid (see, PCT/US00/00207).

Although the foregoing methods and compositions of the invention may beuseful in treated unwanted choroidal neovasculature and therebyameliorating the symptoms of ocular disorders including, for example,AMD, ocular histoplasmosis syndrome, pathologic myopia, angioid streaks,idiopathic disorders, choroiditis, choroidal rupture, overlying choroidnevi, and inflammatory diseases, it is contemplated that the samemethods and compositions may also be useful in treating other forms ofocular neovasculature. More specifically, the methods and compositionsof the invention may likewise be useful at treating and removing orreducing corneal neovasculature, iris neovasculature, retinalneovasculature, retinal angiomas and choroidal hemangiomas.

The invention is illustrated further by reference to the followingnon-limiting examples.

Example 1 Anti-Angiogenesis Factor Potentiates the Effect of PDT onEndothelial Cells

Experiments were performed to determine whether the cytotoxicityresulting from PDT can be potentiated by the addition of ananti-angiogenesis factor. Cells of interest were treated by PDT eitheralone or in combination with an anti-angiogenesis factor and the effecton cytotoxicity of the PDT assessed via a cell proliferation assay.

Bovine retinal capillary endothelial (BRCE) cells (from Patricia A.D'Amore, Schepens Eye Research Institute, Boston, Mass.) and Humanretinal pigment epithelial (RPE) cells (from Anthony P. Adamis,Massachusetts Eye & Ear Infirmary, Boston, Mass.) were grown at 37° C.,5% CO₂ in Dulbecco's modified Eagle's medium (DMEM; Sigma, St. Louis,Mo.), 5% heat-inactivated fetal bovine serum (FBS, Gibco, Grand Island,N.Y.), supplemented with L-glutamine, penicillin, and streptomycin(Gibco Grand Island, N.Y.). Lutetium Texaphyrin (Lu-Tex) was obtainedfrom Alcon Laboratories, Inc. (Fort Worth, Tex.) as a stock solution of2 mg/ml, stable in the dark at 4° C., and used in accordance with themanufacturer's guidelines.

Cell survival was measured using a cell proliferation assay. Briefly,BRCE or RPE cells were plated at a density of 10⁵ cells in DMEM with 5%FBS and incubated at 37° C. in 5% CO₂. After eighteen hours, and ifdesired, recombinant human angiostatin (Calbiochem, La Jolla, Calif.)was added at a concentration of 500 ng/ml. Eighteen hours later, themedium was removed and replaced by 3 μg/ml Lu-Tex in complete media.Thirty minutes later, the cultures were exposed to timed irradiationusing an argon/dye Photocoagulator at 732 nm and laser delivery system(model 920, Coherent Inc., Palo Alto, Calif.). Irradiance was deliveredat a rate of 10 mw/cm² to give a total dose of 5 to 20 J/cm², andirradiation time ranged from 7 to 28 minutes. After irradiation, themedium was removed and replaced with complete medium. Cultures werereturned to the incubator for 7 days, after which cells were dispersedin trypsin, counted in a masked fashion, and the surviving fractiondetermined. The results, reported as the mean of triplicate ±SD, aresummarized in Table 1. Cultures were photographed at various timesfollowing Lu-Tex/PDT using a 16×-0.32 numeric aperture on a phasecontrast inverted microscope (Diaphot, Nikon, Melville, N.Y.).

TABLE 1 Summary of Cellular Survival (%) as a Function of Treatment*Angiostatin Lu-Tex/PDT Cell followed by Lu- followed by Line Lu-Tex/PDTAngiostatin Tex/PDT Angiostatin BRCE 79.13 ± 4.05 (5 J/cm²) 87.39 ± 5.7655.22 ± 3.65 77.61 ± 3.52 53.17 ± 0.32 (10 J/cm²) 38.11 ± 2.50 67.16 ±3.20 33.34 ± 2.26 (20 J/cm²)  0.90 ± 0.32 32.97 ± 2.20 RPE 94.55 ± 1.60(5 J/cm²) 99.09 ± 0.8  91.84 ± 7.97 59.59 ± 3.56 (10 J/cm²) 56.84 ± 6.6153.47 ± 3.18 (20 J/cm²) 45.83 ± 5.51 *The interactive in vitroanti-endothelial effect of combined treatment with angiostatin andLu-Tex/PDT are greater than additive when compared with the sum ofexpected effects of each treatment alone. The potentiation of Lu-Tex/PDTeffect on BRCE was effective with pre-exposure to angiostatin only. Noeffect of angiostatin was observed on RPE. Data are mean % cellularsurvival ± SD.

In order to assess the effect of combining angiostatin to Lu-Tex/PDT onBRCE cell survival, cells were pre-treated for 18 hours with 500 ng/mlangiostatin after which they were treated with Lu-Tex/PDT at variousfluences. Cellular survival was measured by the 1-week cellularproliferation assay. When exposed to angiostatin alone, theproliferation assay demonstrated a 12.61% killing of BRCE cells at theangiostatin dose used (Table 1). Pre-exposing BRCE cells to angiostatindid not appear to interfere with the subsequent cellular uptake ofLu-Tex. More importantly, the results showed a synergistic cytotoxiceffect of angiostatin and Lu-Tex/PDT on BRCE cells at all fluences used(5, 10 and 20 J/cm²), consistently exceeding the cytotoxicity resultingfrom Lu-Tex/PDT alone, angiostatin alone or the arithmetic sum of theirrespective toxicity's (Table 1, FIG. 1A). Controls consisted of cellsexposed to light only because no dark toxicity was observed at theconcentration of Lu-Tex used. Furthermore, it was observed thatangiostatin was not effective in potentiating the effect of Lu-Tex/PDTif delivered after PDT.

In contrast to the results obtained with BRCE cells, no cytotoxicity wasobserved when human RPE cells were treated with human angiostatin, andno interactive killing was observed following exposure to angiostatinand Lu-Tex/PDT (FIG. 1B, Table 1). When combined with angiostatin,Lu-Tex/PDT had a lethal dose (LD₁₀₀) of 20 J/cm² for BRCE cells whereasLu-Tex/PDT alone required 40 J/cm² to achieve the same effect on BRCEcells. Previous studies showed that at fluences of 20 and 40 J/cm², RPEcell survival is about 43% and 21%, respectively.

The data show a specific anti-proliferative effect of angiostatin onBRCE cells as demonstrated by the reduction in cell number in a 1-weekproliferation assay. In contrast, no effect of angiostatin was observedon RPE cells. Accordingly, BRCE cells appear to be another endothelialcell line, along with bovine adrenal cortex-microvascular cells, bovineadrenal cortex capillary cells, bovine aortic cells, human umbilicalvein cells and human dermal microvascular endothelium cells (Mauceri etal. (1998) NATURE 394: 287-291, Lucas et al. (1998) BLOOD 92: 4730-41),that is specifically targeted by angiostatin. In this study, BRCE cellswere used a representative capillary endothelial line of the posteriorsegment to test the anti-angiogenic effect of angiostatin. The findingthat angiostatin induces apoptosis in BRCE cells suggests that celldeath might contribute to the overall reduction of cell number. However,little is known concerning the exact anti-angiogenic mechanism ofangiostatin (Lucas et al. (1998) BLOOD 92: 4730-4741).

In summary, the studies show that Lu-Tex/PDT and angiostatin havecombined cytotoxic effects on retinal capillary endothelial cells, butnot on pigment epithelial cells. However, when angiostatin wasadministered after PDT, the combination did not potentiate the effectsof PDT. In the combination of angiostatin before Lu-Tex/PDT, a fluenceof 20 J/cm² sufficed to achieve nearly 100% mortality of BRCE. In theabsence of angiostatin, a light dose of 40 J/cm² was required to achievethis level of cytotoxicity. At the light dose of 20 J/cm², RPE cellsurvival post-PDT was improved by 20%. The results thus support thepotential of combining angiostatin with Lu-Tex/PDT to improve CNVeradication and to decrease deleterious effects on the RPE.

Example 2 Cellular Morphology Following PDT with Anti-Angiogenic Factor

Experiments were performed to establish how PDT effects the cellularmorphology of BRCE and RPE cells. The cells were treated and exposed toPDT either alone or in combination with angiostatin as described inExample 1. Although cells appeared severely damaged immediately afterPDT, subsequent recovery occurred in certain circumstances. One weekafter PDT, some cells disappeared while those that remained regainedtheir spindle shape and their ability to attach.

In BRCE cells that were first primed with angiostatin followed by PDT,widespread and massive cell death was observed at one week. Onlyremnants of cells and densely refractive bodies of dying cells wereobserved floating in the medium. Particles were recovered and placed infresh complete media but none showed any sign of reattachment orproliferation onto a new dish. The combination of angiostatin andLu-Tex/PDT, therefore, appears to be lethal to BRCE under the conditionsused.

Control BRCE cells and RPE cells which were treated with angiostatinalone for 18 hours continued to proliferate and reached confluence. Noadditive effect of angiostatin to Lu-Tex/PDT was observed in RPE cells.RPE cells subjected to Lu-Tex/PDT alone or with angiostatin appearedunchanged as evidenced by their morphology.

Example 3 Caspase 3-Like (DEVD-ase) Activation in BRCE and RPE FollowingPDT

In order to investigate the role of apoptosis in Lu-Tex/PDT mediatedcell death in BRCE and RPE, the activation of Caspase 3-like (DEVD-ase)protease, a hallmark of apoptosis (Nicholson (1997) TIBS 22: 299-306),was monitored. The kinetics of activation were measuredspectrofluorometrically by assaying the hydrolysis of a substrate thatcan be cleaved only by the caspase 3-like protease family members.

Various times after Lu-Tex/PDT, 10⁶ cells were collected bycentrifugation, and the washed cell pellet resuspended in 500 μl ofice-cold lysis buffer (pH 7.5) containing 10 mM Tris, 130 mM NaCl, 1%Triton X-100, 10 mM NaF, 10 mM NaPi, 10 mM NaPPi, 16 μg/ml benzamidine,10 μg/ml phenanthroline, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 10μg/ml pepstatin and 4 mM 4(2-aminoethyl)-benzene-sulfanyl fluoridehydrochloride (AEBSF). Cellular lysates were stored in aliquots at −84°C. for later protease activity assay or Western blot analysis. A proteinassay (Coomassie Plus protein assay (Pierce, Rockford, Ill.) with abovine serum albumin (BSA) standard was used to assay proteinconcentration in cell extract.

In order to measure protease activity, aliquots containing 50 μg ofcellular protein were incubated with 14 μM (final concentration N-acetyl(Asp-Glu-Val-Asp (SEQ. ID NO: 3)-(7-amino-4-trifluoromethly coumarin)(Ac-DEVD-AFC); (Pharmingen San Diego, Calif.) in 1 ml protease assaybuffer pH 7.2 (20 mM piperazine-N-N′-bis(2-ethanesulfuric acid) (PIPES),100 mM NaCl, 10 mM dithiothreitol (DTT), 1 mM EDTA, 0.1% (w/v)3-[(3-Cholamidopropyl) dimethyl ammonio]-1-propane sulfonate (CHAPS),and 10% sucrose) at 37° C. for 1 hour. Fluorescence was measured using aPerkin-Elmer MPF44A spectrofluorometer (λ_(excitation), 400 nm;λ_(emission) 505 nm). Cellular protein served as the blank. Results werecompared with a standard curve constructed with AFC (Sigma, St. Louis,Mo.) and are shown in FIG. 2.

FIG. 2 illustrates the time course of Ac-DEVD-AFC cleavage afterLu-Tex/PDT at three different light doses in BRCE and RPE. FIGS. 2A, 2Band 2C represent data generated using light does of 10, 20 and 40 J/cm²,respectively. The results show a rapid elevation of caspase 3-likeactivity immediately after Lu-Tex/PDT—as early as 10 min post-Lu-Tex/PDTand peaking at 40 min—in both BRCE and RPE cells and at all doses used.The rate of entry into apoptosis was time and dose-dependent in eachcell line. However, the amount of caspase 3-like activation was alwayssignificantly higher in BRCE cells compared to RPE cells. Furthermore,whereas at 10 J/cm² and 20 J/cm², the amount of caspase 3-likeactivation increased by about 50% in BRCE cells as compared to RPEcells; at 40 J/cm² (equivalent to the LD₁₀₀ for BRCE cells), the levelsin BRCE were 5-fold those in RPE cells.

In order to examine the effect of combining angiostatin and Lu-Tex/PDTon DEVD-ase activation in BRCE cells, cells were treated withangiostatin alone, Lu-Tex/PDT alone and angiostatin/Lu-Tex/PDT,following which caspase 3-like activity was assayed as described above.The results are summarized in FIG. 3. Fluences of 20 and 40 J/cm² wereused, corresponding to LD₁₀₀ of combination angiostatin/Lu-Tex/PDT andLu-Tex/PDT alone respectively. Results demonstrated that the combinationof angiostatin/Lu-Tex/PDT induced a statistically significant increaseof caspase 3-like activity as compared to Lu-Tex/PDT alone using afluence of 20 J/cm² (FIG. 3). However, while both Lu-Tex/PDT (40 J/cm²)and the combination of angiostatin/Lu-Tex/PDT (20 J/cm²) resulted in100% lethality of BRCE cells; Lu-Tex/PDT (40 J/cm²) resulted inincreased levels of caspase 3-like activity as compared toangiostatin/Lu-Tex/PDT (20 J/cm²). As in the case of BRCE cells treatedwith Lu-Tex/PDT alone, the rate of entry into apoptosis of BRCE cellstreated with combination of angiostatin/Lu-Tex/PDT was time-dependent.Nevertheless, the time courses differed significantly in that theinduction of caspase 3-like activation occurred abruptly and morerapidly as a result of angiostatin/Lu-Tex/PDT, peaking at 30 minutes andreaching minimum levels at 90 minutes post-treatment.

Example 4 Modulation of Bcl-₂ Family Members in BRCE and RPE Cells afterLu-Tex/PDT

In order to evaluate the expression of Bcl-2 family members in BRCE andRPE cells after Lu-Tex/PDT, BRCE and RPE cells were subjected toLu-Tex/PDT and the resultant cellular lysates subjected to Western blotanalysis for detection of the anti-apoptotic Bcl-2, Bcl-x_(L) markers,and the pro-apoptotic Bax and Bak markers.

Cell lysates were produced as described in Example 3. Sodium dodecylsulfate-polyacrylamide gel electrophoresis of proteins was performedwith 12% SDS-polyacrylamide gels. All samples were boiled in denaturingsample buffer, and equal amounts of proteins were loaded per lane.Proteins were separated at room temperature under reducing conditions at120 V. Western blot transfer of separated proteins was performed at roomtemperature, using polyvinylidene fluoride membranes at 50 mA for 1 hr.To verify equal protein loading, blots were stained with 0.1% ponceaured (Sigma) diluted in 5% acetic acid. Afterwards, blots were blockedfor 1 hr in Tris buffered saline (TBS; 10 mM Tris-HCl, pH 7.5, and 150mM NaCl) containing 5% non-fat dried milk. Next, the membranes wereprobed with an appropriate dilution (1:250 to 1:1000) of primaryantibody in TBS containing 2.5% non-fat dried milk for one and a halfhours. Mouse polyclonal antibodies against Bcl-2, Bcl-x_(L), Bax and Bakwere purchased from Pharmingen. After incubation with primary antibody,the blots were washed for 30 minutes with frequent changes of TBS,blocked in 1% non-fat dried milk in TBS for 30 minutes, followed byincubation in a peroxidase-coupled secondary antibody for 1 hour in TBScontaining 1% non-fat dried milk. The blots were washed for 1 hour withfrequent changes of TBST (TBS, 0.1% Tween). Immunoblot analysis wasperformed using enhanced chemiluminescence plus Western blottingdetection reagents (Amersham, Pharmacia Biotec Piscataway, N.J.)followed by exposure to x-ray film (ML Eastman Kodak, Rochester, N.Y.).

Results showed a differential expression of members of Bcl-2 familymembers in BRCE and RPE cells. Specifically, Bcl-2 and Bax were detectedin BRCE cells whereas Bcl-x_(L) and Bak were detected in RPE cells(Table 2). After Lu-Tex/PDT at LD₅₀, downregulation of Bcl-2 andupregulation of Bax was observed in BRCE cells resulting in an increaseof the cellular ratio of Bax to Bcl-2 protein. In RPE cells, there wasan upregulation of both Bcl-x_(L) and Bak up to 4 hours post-PDT, afterwhich, Bcl-x_(L) levels reached a plateau, and Bak level started todecline. The upregulation of Bax in BRCE cells appeared to bedose-dependent, however, the upregulation of its pro-apoptoticcounterpart Bak in RPE exhibited dose-dependence only until 20 J/cm²;after which it began to decline.

Lu-Tex/PDT induced caspase 3-like activation in both BRCE and RPE cellsin a dose- and time-dependent fashion, suggesting that apoptosis is amediator of Lu-Tex/PDT cytotoxicity in these cell lines. Furthermore,the data indicate that Lu-Tex/PDT induced apoptosis in BRCE cellsthrough the modulation of Bcl-2 and Bax in a dose- and time-dependentfashion, and in RPE cells through the modulation of Bcl-x_(L) and Bak.As a result, Lu-Tex/PDT may cause different modes of death in each ofthe different cell types.

TABLE 2 Summary of Immunodetection of Bcl₂ Family Members in BRCE andRPE Cells Cell Line Bcl₂ family member BRCE RPE Bcl₂ + − Bcl-x_(L) − +Bax + − Bak − + Detectable (+) or undetectable (−).

After incremental PDT doses, the pro-apoptotic Bak was upregulated inRPE cells until 20 J/cm² following which Bak levels started decliningdespite an increase of PDT dose to 40 J/cm². Without wishing to be boundby theory, it is possible that a protective survival response is mountedin RPE cells at these lethal doses to counteract the apoptotic trigger.Such a hypothesis is further supported by the histologic evidence of RPEcells recovery post-PDT in vivo (Kramer et al. (1996) OPHTHALMOLOGY103(3): 427438, Husain et al. (1999) INVEST OPHTHALMOL VISL SCI. 40:2322-31) and by reports from other investigators showing thatoverexpression of anti-apoptotic Bcl-₂ family members render cellspartially resistant to PDT (He et al. (1996) PHOTOCHEMISTRY ANDPHOTOBIOLOGY 64: 845-852) and inhibits the activation of caspase-3 afterPDT (Granville et al. (1998) FEBS 422: 151-154).

The data show that the combination of angiostatin to Lu-Tex/PDT in BRCEcells resulted in an increase in DEVD-ase activity compared with a samedose of Lu-Tex/PDT applied alone. This suggests that the potentiatingaction of angiostatin on the effect of Lu-Tex/PDT in BRCE cells proceedsthrough apoptosis. However, the time course of caspase 3-like activityfor angiostatin/Lu-Tex/PDT differed from that of Lu-Tex/PDT alone inthat it proceeded faster without latency and peaked as soon as 20minutes after Lu-Tex/PDT. The latter may be explained on the basis thatperhaps the apoptotic cascade was already primed by pre-incubation withangiostatin first, and thus the application of Lu-Tex/PDT benefited froman already lowered threshold of activation to rapidly amplify theapoptotic response. However, this does not exclude the possibility ofthe interplay of more than one apoptotic pathway, especially since PDTis known to initiate cytotoxicity through the generation of reactiveoxygen species (Weishaupt et al. (1976) supra) whereas angiostatin wasrecently shown to act on human endothelial cells by binding to theα-subunit of adenosine triphosphate synthase present on the cell surface(Moser et al. (1999) PROC NATL ACAD SCI USA 96: 2811-2816). Furthermore,whereas angiostatin/Lu-Tex/PDT (20 J/cm²) resulted in a 100% lethalityof BRCE cells as did Lu-Tex/PDT (40 J/cm²) alone, the levels of DEVD-aseactivation were significantly higher in the former regimen. Thissupports the theory that Lu-Tex/PDT and Angiostatin/Lu-Tex/PDT operatethrough different apoptotic pathways in BRCE cells.

Example 5 Targeted Delivery of Photosensitizer to the ChoroidalNeovasculature

It is contemplated that a photosensitizer can be directed to the CNVendothelium by coupling the photosensitizer to a neovascular endotheliumbinding moieties in order to increase the efficacy and lower thetoxicity of PDT. Several targeting molecules may be used to targetphotosensitizers to the neovascular endothelium. The α-v integrins, inparticular α-v β-3 and α-v β-5 integrins, appear to be expressed inocular neovascular tissue, in both clinical specimens and experimentalmodels (Corjay et al. (1997) supra; Friedlander et al. (1995) supra).Cyclic peptide antagonists of these integrins have been used to inhibitneovascularization in experimental models (Friedlander et al. (1996)PROC. NATL. ACAD. SCI. USA 93:9764-9769). A peptide motif ACDCRGDCFC(SEQ ID NO: 2)—also called RGD-4C—was identified that selectively bindsto human α-v integrins and accumulates in tumor neovasculature moreeffectively than other angiogenesis targeting peptides (Arap et al.(1998) NATURE 279:377-380). Another potential targeting molecule is anantibody for vascular endothelial growth factor receptor (VEGF-2R).Clinical and experimental evidence strongly supports a role for VEGF inocular neovascularization, particularly ischemia-associatedneovascularization (Adamis et al. (1996) ARCH. OPHTHALMOL. 114:66-71;Tolentino et al. (1996) ARCH. OPHTHALMOL. 114:964-970; Tolentino et al.(1996) OPHTHALMOLOGY 103:1820-1828). Antibody to the VEGF receptor(VEGFR-2 also known as KDR) can be expected to bind preferentially toneovascular endothelium.

Experimental Design

The photosensitizer Verteporfin (QLT Phototherapeutics, Inc., VancouverBC) or Lutetium Texaphryin (Alcon Laboratories, Fort Worth, Tex.) willbe coupled to a targeting moiety, for example, an RGD-4C peptide, or ananti-VEGF receptor antibody using standard coupling chemistries. Thespectral characteristics (emission & excitation) of the resultingphotosensitizer complex can be measured in vitro. Subsequently, in vitrostudies can be carried out using BRCE and RPE cells, to assess cellularuptake and phototoxicity following PDT. Experiments may address PDTtreatment parameters including optimal timing as well as drug and lightdosimetry for selective phototoxicity in vitro. Then, the efficacy andselectivity of PDT using the bound photosensitizer in vivo in the ratmodel of CNV can be tested. The results of PDT with photosensitizercomprising the targeting molecule may then be compared to the results ofPDT with the same photosensitizer lacking the targeting molecule.

CNV can be induced in animals using a krypton laser, and documented bydigital fundus fluorescein angiography. More specifically, the laserinjury model in the rat is a modification of a similar model in themonkey (Dobi et al. (1989) ARCH. OPHTHALMOL. 107:264-269; Ryan (1982)ARCH. OPHTHALMOL. 100:1804-1809; Tobe et al. (1994) J. JPN. OPHTHALMOL.SOC. 98:837-845). Briefly, 5-6 high intensity krypton laser burns (100μm spot size, 0.1-second duration, 160 mW) can be placed in aperipapillary fashion. CNV as evidenced by hyperfluorescence and lateleakage can be documented using digital fluorescein angiography and isexpected to develop in at least 60% of the lesions within 2-3 weeks oflaser injury.

PDT can then be performed over areas of CNV and normal choroid and theeffects assessed angiographically and histologically. More specifically,PDT may be carried out using tail vein injection of the photosensitizereither containing or lacking a targeting molecule, followed by laserirradiation of the treatment area. PDT may also be applied to areas ofCNV in one eye and to areas of normal choroid in the fellow eye.Photosensitizer and laser parameters will be based on previousexperiments using Verteporfin and Lu-Tex in the monkey model, as well assome preliminary dosimetry in the rat model.

The efficacy of PDT can be assessed as follows:

(a) Efficacy of CNV closure. Effective closure of CNV can be assessed bythe absence of leakage from CNV via fluorescein angiography 24 hoursafter PDT. This methodology has been well established in the laserinjury in the monkey. Histopathology can be carried out using lightmicroscopy.(b) Selectivity of Effect. Since CNV in this model develops in an areaof laser injury, it is difficult to assess the effects of PDT on retinaand choroid when areas of CNV are treated. Therefore, to demonstrate theselectivity of PDT to CNV, PDT may also be applied to areas of normalretina and choroid and a published histopathologic grading scheme usedto quantify damage to RPE, photoreceptors, retinal and choroidal vessels(Kramer et al. (1996) OPHTHALMOLOGY 103:427-438).(c) Comparison of the Effects of PDT versus combined PDT regimens. Theeffects of PDT may be compared between groups of CNV animals treatedwith PDT using photosensitizer alone, and groups receiving modified PDT(i.e. targeted photosensitizer). PDT may be applied to the CNV andnormal areas. First, it may be determined if CNV closure occurs at thesame light dose (fluence J/cm²) using the modified PDT as with PDTalone. Then, at the identified light dose, the effects of modified PDT,and PDT alone, on normal choroid may be compared. As an example, usingthe targeted photosensitizer, one may be able to achieve closure of CNVat a lower fluence than with unbound photosensitizer, and at thisfluence one may find much less damage to the RPE in normal areas treatedwith PDT using targeted photosensitizer.

Example 6 Combined Effects of Targeted Pro-Apoptotic Peptides and PDTfor Choroidal Neovascularization Treatment

Experiments have shown that PDT induces cell death in endothelial cellsby apoptosis and that its toxicity towards the RPE also proceeds byprogrammed cell death. Different apoptotic pathways appear to betriggered by PDT in BRCE and RPE cells. It is contemplated that byspecifically priming the apoptotic machinery of neovascular capillaryendothelial cells prior to PDT it may be possible to increase theirsensitivity to PDT. This approach may reduce the light dose (fluence)required to achieve CNV closure and thereby decrease the effect on thesurrounding cells such as RPE cells.

Studies have shown the efficacy of targeted pro-apoptotic peptides inanti-cancer activity in significantly reducing the tumor size (Ellerbyet al. (1999) supra). These targeted pro-apoptotic conjugates werecomprised of two functional domains: an antimicrobial peptide(KLAKLAKKLAKLAK; SEQ ID NO: 1) with low mammalian toxicity and anangiogenic homing peptide (RGD-4C). The antibacterial peptidepreferentially disrupts prokaryotic membranes and eukaryoticmitochondrial membranes rather than eukaryotic plasma membranes (Ellerbyet al. (1999) supra). Thus the chimeric peptide, therefore, may have themeans to enter the cytosol of targeted cells, where it inducesmitochondrial-dependent apoptosis. Endothelial cells primed with theseconjugates are expected to be more susceptible to PDT.

Experimental Design

Peptides of interest will first be tested in vitro in BRCE and RPE cellsto ascertain specificity and efficacy. Then, the pro-apoptoticpeptide/PDT regimen may be assessed in vitro, and then compared with PDTalone and peptide alone in both BRCE and RPE cells. BRCE and RPE cellsmay be grown using standard tissue culture techniques. The ApoAlertassay kit (Clonetech) may be used to assay for caspase-3 like activityin cells post-treatment. This colorimetric assay follows the chromophorep-nitroanilide (pNA) arising from cleavage of the substrate DEVD-pNA.DEVD-pNA is a known substrate for active caspase-3 and can be added tocellular extracts prepared at different time points after treatment, andsamples can be analyzed to assess caspase-3 activity.

Thereafter, experiments may be carried out to test the efficacy andselectivity of targeted pro-apoptotic peptide in vivo in the rat modelof CNV. Targeted pro-apoptotic peptides may be injected intravenously 4hours prior to PDT. PDT may be performed over areas of CNV and in normaleyes, comparing the effect on CNV closure of PDT alone with PDT afterpro-apoptotic peptide, and comparing the selectivity in normal choroidas described in Example 5.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes which come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

INCORPORATION BY REFERENCE

Each of the patent documents and scientific publications disclosedhereinabove is expressly incorporated herein by reference.

1-19. (canceled)
 20. A method of treating unwanted choroidalneovasculature in a mammal, the method comprising the steps of: (a)administering to the mammal, an apoptosis-modulating factor in an amountsufficient to permit an effective amount to localize in the choroidalneovasculature or tissue surrounding the choroidal neovasculature; (b)administering to the mammal an amount of photosensitizer sufficient topermit an effective amount to localize in the choroidal neovasculature;and (c) irradiating the choroidal neovasculature with laser light suchthat the light is absorbed by the photosensitizer so as to occlude thechoroidal neovasculature.
 21. The method of claim 20, wherein the mammalis a primate.
 22. The method of claim 21, wherein the mammal is a human.23. The method of claim 20, wherein the factor is administered to theprimate before administration of the photosensitizer.
 24. The method ofclaim 20, wherein the photosensitizer is an amino acid derivative, anazo dye, a xanthene derivative, a chlorin, a tetrapyrrole derivative, ora phthalocyanine.
 25. The method of claim 20, wherein thephotosensitizer is lutetium texaphyrin, a benzoporphyrin, abenzoporphyrin derivative, a hematoporphyrin or a hematoporphyrinderivative.
 26. The method of claim 20, wherein the apoptosis modulatingfactor induces or represses apoptosis.
 27. The method of claim 26,wherein the factor is a peptide.
 28. The method of claim 27, wherein thepeptide selectively binds to neovasculature.
 29. The method of claim 27,wherein the peptide induces apoptosis in endothelial cells.
 30. Themethod of claim 29, wherein the peptide comprises an amino acid sequencecomprising, in an N- to C-terminal direction, KLAKLAKKLAKLAK (SEQ. ID.NO 1).
 31. The method of claim 30, wherein the peptide further comprisesan RGD-4C peptide sequence.
 32. (canceled)
 33. The method of claim 20,wherein the level of cell damage to the choroidal neovasculaturerelative to the tissue surrounding the choroidal neovasculatureresulting from steps (a), (b) and (c) is greater than that resultingfrom steps (b) and (c) alone.